This invention relates to mammalian artificial chromosomes (MACs) that can replicate autonomously, can be stably maintained extra-chromosomally and can be transmitted efficiently in mammalian cells. The invention includes methods to construct, modify and stably maintain yeast artifiical chromosomes (YACs) in yeast cells which have the potential ability to form MACs when introduced into mammalian cells.
Constructing a mammalian artificial chromosome (MAC) carrying the known functional elements required for chromosome replication, stable extra-chromosomal maintenance and segregation in a manipulatable form will be of great value not only for basic studies concerning the organization and function of mammalian chromosomes but also as a vector to introduce DNA segments (genes) of interest to test their functions in mammalian cells or bodies, since the genes carried by MACs will neither be subject to variable expression due to integration position effects nor cause unpredictable insertion mutation in the host chromosomes. Furthermore, a MAC will have the capacity to accommodate a DNA segment having a size in the megabase range, wherein an entire large gene or group of genes and regulatory elements could be included. For these reasons, MACs will offer exciting alternative vectors to currently existing vectors for somatic gene therapy, because frequently used infectious vectors derived from viruses are either integrated randomly into host chromosomes or exist extra-chromosomally but only transiently. Besides, these vectors are able to carry only short DNA segments. A new way to generate transgenic mice will be provided by the invention of MACs, if the stability of MACs during meiosis is established in mammalian development. However, the construction of a MAC has not yet been achieved due to technical difficulties (Willard, Proc. Natl. Acad. Sci. 93, 6874-6850, 1996).
Yeast artificial chromosomes (YACs) has been constructed (Burke et al., Science, 236, 806-812, 1987) with three essential DNA elements from the budding yeast, Saccharomyces cerevisiae; namely, an origin of replication or autonomously replicating sequence (ORI or ARS) is required for initiation of DNA replication, telomere sequences (TEL) are required to stabilize and facilitate complete replication of chromosomal ends, and a centromere (CEN) is required for faithful segregation of the sister chromatids after replication. As a result, YACs have become a major tool for cloning large gene segments of complex genomes. Similar to YACs, MACs are believed to be constructable with the three essential elements derived from mammalian genomes. Among the three, telomeres have been isolated from mammalian chromosomes and used for the mammalian chromosome manipulation (Brown et al., Hum. Mol. Genet., 27-1237, 1994; Farr et al., EMBO J., 14, 5444-5454, 1995), but centromeres and the origins of replication for mammalian chromosomes were found to be difficult to isolate because of the unavailability of activity assays.
The present investigators have analyzed the specific structure of the mammalian centromere locus in order to obtain information concerning the essential functional structure of mammalian centromeres. They have found that centromere protein B (CENP-B), which is one of the antigens recognized by anti-centromere antibodies (Moroi et al., Proc. Natl. Acad. Sci. USA, 77,1627-1631, 1980) at centromeres of various mammalian chromosomes, specifically recognizes and binds the 17 bp sequence (CENP-B box) of the centromere satellite DNA (alphoid DNA) in the human genome (Masumoto et al., J. Cell Biol., 109, 1963-1973, 1989; Muro et al., J. Cell Biol., 116, 585-596, 1992). The recognition sequences of CENP-B were found in centromeric satellite DNA of mouse species (Masumoto et al., J. Cell Biol., 109, 1963-1973, 1989; Kipling et al., Mol. Cell. Biol., 15, 4009-4020, 1995) and the consensus sequence of the CENP-B box was established to be 5xe2x80x2-NTTCGNNNNANNCGGGN-3xe2x80x2 (Masumoto et al., NATO ASI Series, vol.H72, Springer-Verlag, 31-43, 1993; Yoda et al., Mol. Cell. Biol.,16, 5169-5177, 1996). They have also demonstrated that a pair of CENP-B sequences form a dimer at the C-terminal and bind to a CENP-B box located in the centromeric satellite DNA at the N-terminal of each CENP-B polypeptide, so that a stable complex, which consists essentially of the dimer protein and the two regions (or strands) of the DNA, was formed (Muro et al., J. Cell Biol.,116,585-596, 1992; Yoda et al., J. Cell Biol., 119, 1413-1427, 1992; Kitagawa et al., Mol. Cell. Biol. 15, 1602-1612, 1995). Further, the investigators studied the location of the CENP-B box in human chromosome 21 (Ikeno et al., Hum. Mol. Genet., 3, 1245-1257, 1994), and found that there are two distinct regions in the alphoid DNA array of human chromosome 21 with regard to the CENP-B box. Specifically, one (xcex121-I) contains a regular series of CENP-B box sequences, and the other (xcex121-II) scarcely contains any CENP-B box sequences, the two regions extend side by side for a stretch of about several megabases, the former is located at a position in the chromosome where the centromere proteins are located and the latter is located at a position slightly shifted towards the short arm of the chromosome (Ikeno et al., Hum. Mol. Genet., 3, 1245-1257, 1994).
The present investigation relates to the DNA region within the CENP-B box. It is an object of the investigation to provide artificial chromosomes derived from the above region that can be stably maintained in the extra-chromosomal region of mammalian cells, especially in human cells, and can be safely transmitted to cells of succeeding generations. The present investigation includes development of methods to construct, modify and stably maintain the precursors of such artificial chromosomes in yeast cells as YACs, which have the potential ability to form mammalian artificial chromosomes when introduced into mammalian cells.
This invention has been achieved by using homologous recombination in yeast cells to determine a method for constructing a yeast artificial chromosome construct comprising a DNA sequence including CENP-B box sequences from the alphoid DNA region of human chromosome 21 that can interact with CENP-B or the centromere protein B, and a segment of the human telomere sequence. Further, the invention involves the determination that when the construct is introduced into a human cell, the construct is replicated autonomously in a human cell and is stably maintained in the cell lineage.
This invention provides a DNA construct comprising a mammalian telomere and a centromere, wherein the centromere has a DNA sequence containing a plurality of copies of the CENP-B box sequence consisting of:
5xe2x80x2-NTTCGNNNNANNCGGGN-3xe2x80x2,
wherein N is any one of A, T, C and G.
In a preferred embodiment of this invention, a DNA construct comprises a mammalian telomere and a centromere, wherein the centromere has a DNA sequence containing a plurality of copies of the CENP-B box sequence designated herein as sequence No. 1.
Preferably, the centromere contains spaced repeats of the CENP-B box sequence.
This invention provides a DNA construct comprising a mammalian telomere and a centromere, wherein the centromere has a DNA sequence containing a plurality of copies of the sequence designated as sequence No. 2 or a sequence derived from sequence No.2 in which one or more nucleotides are added, deleted and/or replaced.
In a preferred embodiment of this invention, the centromere is derived from a human chromosome. The DNA construct further comprises one or more sequences necessary for the DNA construct to multiply in yeast cells. The DNA construct further comprises a sequence encoding a selectable marker gene. The DNA construct is capable of being maintained as a chromosome in a transformed cell with the DNA construct. Preferably, the DNA construct is capable of being maintained as a chromosome in a human cell. Further, the DNA construct is capable of being maintained as a chromosome in a mouse cell.
This invention provides a host cell transformed with the DNA construct. In a preferred embodiment, the host cell is a human cell. Further, the host cell is a yeast cell. Still further, the host cell is a mouse cell.
This invention provides a DNA construct that further comprises the sequence of a gene of interest. The DNA construct further comprises a genome DNA sequence containing a structural region and its regulatory region.
This invention provides a method of homologous recombination comprising the steps of:
(i) producing a recombinant DNA construct in a DNA recombination deficient host cell, wherein the host cell comprises a plasmid for DNA recombination and the recombinant DNA construct is prepared from two or more linear and/or circular DNA sequences that are partially homologous, and
(ii) collecting cells that carry the recombinant DNA construct but do not carry the plasmid.
This invention, which allows a highly efficient intracellular homologous recombination can simplify conventional in vitro recombination based on the use of restriction enzymes, and thus offers a new recombination method whereby a given DNA segment can be formed. Preferably, the host cell is a yeast cell. Further, the recombinant DNA construct is from a yeast artificial chromosome. Still further, one of the DNA sequences is from a yeast artificial chromosome. Additionally, one of the DNA sequences is from a yeast artificial chromosome which has a repetitive DNA sequence.
This invention provides a method of making a yeast artificial chromosome construct comprising the steps of:
(i) producing a first recombinant yeast artificial chromosome having a mammalian telomere and a centromere in a DNA recombination deficient host cell having a plasmid for DNA recombination, wherein the centromere has a DNA sequence containing a plurality of copies of the CENP-B box sequence consisting of:
5xe2x80x2-NTTCGNNNNANNCGGGN-3xe2x80x2,
wherein N is any one of A, T, C and G.
(ii) selecting cells carrying the first recombinant yeast artificial chromosome, but not carrying the plasmid.
(iii) producing a second recombinant yeast artificial chromosome, using the telomeres and the centromere from the first recombinant yeast artificial chromosome in the host cell having a plasmid for DNA recombination, and
(vi) selecting cells carrying the second recombinant yeast artificial chromosome, but not carrying the plasmid.
This invention provides a method of producing a mammalian artificial chromosome comprising the step of:
introducing a DNA construct comprising a mammalian telomere and a centromere into a mammalian cell, wherein the centromere has a DNA sequence containing a plurality of copies of the CENP-B box sequence consisting of:
5xe2x80x2-NTTCGNNNNANNCGGGN-3xe2x80x2,
wherein N is any one of A, T, C and G.
Further, the mammalian cell is a human cell. Further, the mammalian cell is a mouse cell. Still further, the DNA construct is derived from the Saccharomyces cerevisiae xcex17C5hTEL that has been designated as FERM BP 5625.
This invention provides a mammalian artificial chromosome which is produced according to the method of producing a mammalian artificial chromosome of this invention.
This invention provides a method of fragmenting a chromosome comprising of step of:
introducing a first DNA construct comprising a mammalian telomere, a centromere and a DNA sequence that is partially homologous to the chromosome and a second DNA construct comprising a mammalian telomere and a DNA sequence that is homologous to the chromosome into the mammalian cell, wherein the centromere has a DNA sequence containing a plurality of copies of the CENP-B box sequence consisting of:
5xe2x80x2NTTCGNNNNANNCGGGN3xe2x80x2,
wherein N is any one of A, T, C and G.